The invention relates to methods and compositions for the transformation of sorghum, particularly to methods for transformation utilizing Agrobacterium.
Sorghum is one of the most important cereal crops for subsistence farmers in arid and semi-arid portions of Africa, Asia and the Americas. The crop is essential for human life on marginal lands throughout the poorest regions of the world. However, further development of the crop is needed if food production in these areas is to keep pace with increases in population. In developed countries, sorghum is important as a feed crop and as a crop that can be grown on marginal lands as part of a sustainable agroecosystem.
Sorghum is typically the cereal grown in areas where the extremes of high temperature and low soil moisture are unsuitable for maize. In 1991, sorghum was ranked fifth in production for all cereals with 58 million metric tons harvested on 45 million hectares of land. See, Food and Agriculture Organization of the United Nations (1992), FAO Production Yearbook 1991 (FAO, Rome) Volume 45. Sorghum is used primarily as livestock feed in the western hemisphere. The development of hybrid varieties of sorghum in the 1950s contributed substantially to the increase in production in the United States. Presently, sorghum ranks third among the cereals produced in the U.S. and is the preferred crop in areas of low water availability because of its yield stability under drought conditions.
Sorghum is plagued by diseases, especially in higher yielding environments. Many of the diseases are caused by highly variable pathogens. Generally, as yield potential increases, so does the proportion of the crop lost to diseases.
Until recently, genetic improvement of sorghum for agronomic and quality traits has been carried out by traditional plant breeding methods and improved cultural management practices. Advances in tissue culture and transformation technologies have resulted in the production of transgenic plants of all major cereals, including sorghum. To date, key to this transformation was the development of microprojectile bombardment devices for DNA delivery into cells. Microprojectile bombardment circumvented two major constraints of cereal transformation. These constraints are the lack of an available natural vector such as Agrobacterium tumefaciens and the difficulty to regenerate plants when protoplasts are used for transformation. Particle bombardment can target cells within tissues or organs that have high morphogenic potential. However, the use of microprojectile bombardment as a transformation vehicle has its drawbacks. Particularly, with bombardment several copies of the gene to be transferred are often integrated into the targeted genome. These integrated copies have often been rearranged and mutated. Furthermore, the transformation event may not be stable due to the insertion point or means still not an efficient process (Casas et al. (1993) Proc. Natl. Acad. Sci. USA 90:11212-11216).
Agrobacterium, a natural plant pathogen, has been widely used for the transformation of dicotyledonous plants. Agrobacterium remains the most widely used vector for transformation of dicot species. Because monocotyledonous plants are rarely natural hosts for Agrobacterium, they have not been expected to be susceptible to gene transfer mediated by the bacterium.
The advantage of the Agrobacterium-mediated gene transfer is that it offers the potential to regenerate transgenic cells at relatively high frequencies without a significant reduction in plant regeneration rates. Moreover, the process of DNA transfer to the plant genome is defined. That is, the DNA does not normally undergo any major rearrangements, and it integrates into the genome often in single or low copy numbers.
Agrobacterium-mediated transformation involves incubation of cells or tissues with the bacterium, followed by regeneration of plants from the transformed cells via a callus stage. Inoculation of explants has proven to be the most effective means of creating transgenic plants.
Early work with Agrobacterium indicated that the bacterium could transfer T-DNA to monocotyledonous hosts. However, clear evidence of T-DNA integration existed only for asparagus, and even in that case, no transformed plants were produced. Because of the recalcitrant nature of monocots to Agrobacterium infection, other methods, such as particle bombardment, were developed for the transformation of monocots. More recently, the transformation of maize and rice using Agrobacterium has been reported. See, Ishida et al. (1996) Nature Biotechnology 14:745-750; EPA 0672752A1; EPA 0687730A1; and U.S. Pat. No. 5,591,616. Among the factors indicated that affect the efficiency of transformation include the types and stages of maize tissues infected, the concentration of A. tumefaciens, compositions of the media for tissue culture, selectable marker genes, kinds of vectors and Agrobacterium strains, and the maize genotype. The researchers concluded that the main hurdle in transformation may have been the recovery of cells that acquired the T-DNA in their chromosomes. See, Ishida et al. (1996) supra.
While reports indicate that some genotypes of maize and rice can be transformed with Agrobacterium, there is no report of Agrobacterium-mediated transformation of sorghum. While transgenic sorghum plants have been reported following microprojectile bombardment, transgenic plants were obtained only at very low frequencies. Further, inherent characteristics of the sorghum cells make them somewhat unresponsive for transient expression. Casas et al. (1993) Proc. Natl. Acad. Sci. USA 90:11212-11216.
Accordingly, there is needed an efficient method for the transformation of sorghum wherein stable transformation of large inserts can be obtained. That is, there is needed a method for the transformation of sorghum utilizing Agrobacterium.
The present invention is drawn to methods and compositions for the efficient transformation of sorghum. The method involves the use of bacteria belonging to the genus, Agrobacterium, particularly those comprising a super-binary vector. In this manner, any gene of interest can be introduced into the sorghum plant. The transferred gene will be flanked by at least one T-DNA border and present in the transformed sorghum in low copy number.
Transformed sorghum cells, tissues, plants, and seed are also provided. Such transformed compositions are characterized by the presence of T-DNA borders and a low copy number of the transferred gene. The invention encompasses regenerated, fertile transgenic sorghum plants, transgenic seeds produced therefrom, T1 and subsequent generations.